Protein removal agent

ABSTRACT

The present invention provides compositions, methods and kits for the removal of proteins from complex reaction mixtures useful in majority workflows of molecular biology research experiments. More specifically, such compositions, methods and kits are useful in such processes as purification of nucleic acids from biological samples or after their treatment with specific enzymes, when residual enzyme activity in reaction mixture is not compatible with downstream applications.

This application claims priority to co-pending U.S. application Ser. No.13/953,263 filed Jul. 29, 2013; which claim priority to Great BritainApplication Serial No. 1217405.8, filed Sep. 28, 2012, the entirety ofeach of which is incorporated by reference herein in its entirety.

The invention provides compositions, methods and kits for the removal ofproteins from complex reaction mixtures useful in majority workflows ofmolecular biology research experiments. Such compositions, methods andkits are useful in processes such as purification of nucleic acids frombiological samples or after their treatment with specific enzymes, whenresidual enzyme activity in reaction mixture is not compatible withdownstream applications.

Purification of nucleic acids from solutions containing enzymes actingon the nucleic acids and/or other proteinaceous materials is afrequently performed operation in life science research. Removal ofenzymes used in the preceding manipulation stage from nucleic acidssolutions is almost always required before downstream processing.

There are many methods for protein/enzyme removal from reaction mixturesknown in the art. The main parameters important for the user of suchmethods are complete inactivation of any enzyme and denaturation/removalof all proteins from reaction mixture, good yields of nucleic acids,convenience of use, and absence of hazardous materials and safetyconcerns.

One of the first methods used for protein removal from biologicalsolutions/reaction mixtures was liquid partitioning method using organicsolvents. This method described by Kirby (Biochem. J. 66: 495-504 (1957)and Marmur (J. Mol. Biol. 3: 208-218 (1961)), with many latermodifications, uses the difference of partition behavior of nucleicacids and proteins in organic/water two phase systems, where proteinsmove to the organic phase and nucleic acids are collected from theaqueous phase. The aqueous solution of nucleic acids is usuallysequentially partitioned in phenol/water and in chloroform/waterbiphasic systems.

Despite high efficiency of protein removal, this method hasdisadvantages. It is labor- and time-consuming. It is a deemed healthhazard because the organic compounds used are flammable, corrosive,toxic, irritant and carcinogenic. It is a deemed environmental hazardbecause large volumes of non-biodegradable compounds are used.Additional manipulation steps are required to remove organic compounds:washing aqueous phase with diethyl ether, precipitating nucleic acidswith ethanol/acetate, dialysis, etc.; these are time consuming and mayresult in some nucleic acid loss and thus lower yields.

Another known method of protein/nucleic acids separation is based on anadsorption/elution cycle on solid support. This method (Marko et al.,Anal. Biochem. 121 (1982) 382 and later modified by Boom et al. J. Clin.Microbiol. 28 (1990) 495) uses the property of nucleic acids to bind tosilica, e.g., glass milk, glass fibers, diatomaceous earth, particulateand membranous silica, zeolites, etc., in the presence of alcohol andeither chaotropic or kosmotropic (Invitek GmbH EP 1135479B1) agents.

This procedure is usually embodied in the form of spin-columnpurification where the solid phase active substance, silica, is packedinto a small column through which the flow of liquid phase is achievedby applying centrifugal force. A series of binding, washing and elutionsteps is applied.

This method is more convenient for the user because no irritant andfoul-smelling substances are required, and reagents are used in lowerquantities, are less hazardous, and are more environment-friendly.However, this method has the following disadvantages: it consists ofmultiple manipulations and centrifugation steps that requirecentrifugation equipment compatible with spin column format, thecomparatively low selectivity of silica requires the use of columntechnology, the yield of nucleic acids is limited by the adsorptioncapacity of silica layer as well as by the dead volume of the samelayer, it uses a multistep procedure requiring time and skilled labor,and certain impurities, e.g., RNases, may survive the procedure and maybe detrimental to downstream applications.

Another widely used method of protein elimination from reaction mixturesis enzyme inactivation by heat. This method is simple and is applicableif the only substance interfering with downstream workflow is the activeform of the enzyme. Enzyme classes that are most often eliminated fromreaction mixtures by heat inactivation are deoxyribonucleases(non-specific deoxyribonucleases and restriction enzymes). The mostfrequently used non-specific nuclease is pancreatic DNase I which iseasily inactivated by heat. However, Ca²⁺ and Mg²⁺ ions, when available,interfere with thermoinactivation (Chen and Liao, Protein Peptide Lett.13: (2006) 447), and addition of chelates is required before heattreatment (Huang et al., BioTechniques 20 (1996) 1012; Silkie et al., J.Microbiol. Meth. 72 (2008) 275). However, even thermoinactivated DNase Iwill partially regain its activity during downstream procedures aftercontact with Ca²⁺ or Mg²⁺ ions (Bickler et al., BioTechniques 13 (1992)64). If the nucleic acid to be purified was RNA, it will be degraded byheat as well, although less if chelators and reducing agents werepresent in solution, and the thermoinactivation conditions must bethoroughly optimized to maximize the yield of RNA (Huang et al., op.cit.]. There are many deoxyribonucleases (both non-specific andrestriction enzymes) that are very resistant to elevated temperatures.Most ribonucleases are impossible to inactivate by heat at all, and manyenzymes regain their activity after thermoinactivation.

Although heat inactivation has advantages of convenience, speed, andsimplicity, it has the following disadvantages: it is suitable only forcertain contaminating proteins, e.g., for DNase I; it does notphysically separate contaminating protein species from the workingsolutions comprising nucleic acids, but only stops or substantiallyreduces enzymatic activity; it may cause deleterious side effects, e.g.,reactivation of thermoinactivated enzymes, degradation of target nucleicacid, and various physical and chemical processes such as depurinizationof DNA, formation of precipitates etc.; and enzyme inactivation ispartially reversible.

Another method for removal of undesirable enzymatic activity fromreaction mixtures is inactivation of enzymes by chemical substancesleaving the inactive species physically in the solution. However, themajority of chemical substances that inactivate enzymes exhibit lowspecificity towards specific enzymatic activity, and thus the reactionmixture must be re-purified prior to performing subsequent reactions.These substances also often modify not only enzymes but nucleic acids aswell, e.g. diethyl pyrocarbonate modifies adenine residues. The notableexception is mammalian ribonuclease inhibitor protein (Blackburn et al.,J. Biol. Chem. 252 (1977) 12488) which inhibits ribonucleases from RNaseA class.

The above described method has the advantage of convenience, speed andsimplicity, but has the following disadvantages: it is suitable only forcertain contaminating materials, e.g., placental RNase inhibitor usedfor RNase A; it does not physically separate contaminating species fromthe reaction mixture but only stops their action; it introduces foreignchemical substances that must be purified from the reaction mixturebefore downstream workflow; chemical substance may display deleteriousactivity against nucleic acids; and some substances, e.g., diethylpyrocarbonate, are toxic, irritant, carcinogenic, etc.

Several suppliers offer commercial products for enzyme/protein removalfrom reaction mixtures based on the principle of impurities adsorptiononto a solid support, leaving nucleic acids in the liquid phase. Thisclass of methods (McCormick, Analyt. Biochem. 181 (1989) 66; U.S. Pat.Nos. 4,923,978; 7,264,932; QuickClean reagent (Clontech) exploits a verysimple solid-phase extraction procedure enabled by the extremely stronginteraction between specialty adsorbents and proteins to be removed. InMcCormick, a phenol silica is prepared for this purpose. In contrast,U.S. Pat. No. 4,923,978 describes a different process for purifyingnucleic acids. A solid phase extraction material is prepared from acommercially available silica by removing polyvalent cationic materialfrom the surface thereof. This is achieved by acid washing to produce apure native silica surface layer to prevent nucleic acid adsorptionthereto. This solid phase was able to bind enzymes with sufficientstrength even in static conditions without significant binding ofnucleic acids.

The main disadvantages characteristic for this class of methods are: theadsorbent must be thoroughly designed for a given enzyme; residualactivities of enzymes after removal may be still high enough tointerfere with downstream workflow; non-specific nucleic acid sorptiononto solid support may negatively affect its yield after treatment; andthe method is more prone to user error than most alternatives.

A need thus exists for efficient universal protein removal from reactionmixtures reagent that exhibit the following characteristics: residualenzyme activity after their removal is sufficiently low to avoidinterference with downstream workflow; nucleic acid yields are almostthe same before and after treatment with the reagent; the reagent isconvenient to use; and the reagent is free from hazardous materials andhas no safety concerns. Such method must be quick and simple, andcapable of total removal of enzymes from various reaction mixturesleaving nucleic acids in biologically active state without introducingforeign compounds interfering with downstream procedures.

In a first aspect, the invention provides use of a solid phase having asurface that comprises a functionalized silica for removal of proteincontaminants from a solution containing target nucleic acid, where thefunctionalized silica bears anionic or neutral substituent groups thatare (i) polar, other than phenol, and/or (ii) comprise a C₁ to C₃ alkylchain.

The invention provides an improved and simplified method and compositionfor removal of protein contaminants from solutions containing useful,i.e., target, nucleic acids. These solutions may be complex mixturesoriginating from biological samples in the workflow of nucleic acidssample preparation for further analysis using well known methods such asPCR, qPCR, cloning, or next generation sequencing workflow. The solidphase may be used for removal of protein contaminants such as nucleicacid enzymes. These may be enzymes acting on nucleic acids, such asDNA/RNA polymerases, non-specific deoxy- and ribonucleases such as DNaseI, restriction endonucleases, RNase A, etc., phosphatases, ligases fromreaction mixtures in routine laboratory workflow prior to transferringnucleic acid into the next reaction step. The protein removal effect inthe present invention is achieved by the solid phase.

The surface of the solid-phase comprises a functionalized silica thatmay be present as a coating on a substrate which is a non-silicasubstrate, or the entire solid phase may comprise silica. The solidphase has at its surface substituent groups that may be polarsubstituent groups and/or weakly hydrophobic substituent groups. Thesubstituent groups may be anionic or neutral substituent groups that are(i) polar, other than phenyl, and/or (ii) comprise a C₁ to C₃ alkylchain. Polar neutral substituent groups are typically dipoles, which maybe immobilized to the solid phase by short hydrophobic handles thattypically comprise C₁ to C₃ alkyl chain. Anionic substituent groupsinclude sulfonate or alkanoyl groups that may be immobilized to thesolid phase by a hydrophobic handle such as C₁ to C₃ alkyl chain.

The absence of cationic substituent groups and phenolic substituentgroups inhibits or prevents nucleic acid binding to the solid phase,which is undesirable and may lead to reduced nucleic acid yields whenthe solid phase is used.

In one embodiment the substituent groups are selected from cyanoalkylgroups (CN—(CH₂)_(n)—) where n is an integer that is at least 3; shortchain alkyl groups (CH₃—(CH₂)_(m)—) where m is 0, 1 or 2; sulfoalkylgroups (HSO₃—(CH₂)_(n)—) where I is an integer in the range 2 to 6,alkanoyl groups (CH₃—(CH₃)_(p)—CO—O—) where p is 0, 1 or 2; or a diol((OH)₂—CH—(CH₂)_(r)—), or hydroxyl OH—(CH₂)_(r)— where r is zero or aninteger in the range 1 to 5, or mixed diol CH₂OH—CHOH—(CH₂)_(t)—; orcyclohexyldiol C₆H₉(OH)₂—(CH₂)_(t)—; or alkylhalides Hal-(CH₂)_(t)—; ortosylhalides SO₂Hal-C₆H₄—(CH₂)_(t)—; or tosyl SO₃H—C₆H₄—(CH₂)_(t)—; oralkylphenylhalides Hal-(CH₂)_(u)—C₆H₄—(CH₂)_(t)— where Hal is halogen, tis 2, 3, or 4, and u is 0, 1, or 2.

In one embodiment cyanoalkyl groups are cyanopropyl groups. In oneembodiment alkanoyl groups are acetyl groups.

The solid phase typically comprises particles that may be approximatelyspherical and may have a diameter in the range of 3 μm to 15 μm. In oneembodiment the particles have pores with a diameter of from 10 nm to 100nm. Porous silica particles provide a relatively high surface to volumeratio that facilitates binding of the protein contaminants to theparticles.

In one embodiment the surface of a solid phase further comprises smallsubstituted silane groups —O—Si—R₃ where R is C₁ to C₃ alkyl groups ormixture thereof. These groups may be formed by capping any free silanolgroups present on the surface of the solid phase, e.g., after the silicasurface has been functionalized with the bulky surface substituentgroups discussed above. This prevents unwanted nucleic acid binding.

Removal of protein contaminants is carried out in the presence of a polyacid that comprises a polycarboxylic acid, a polyphosphonic acid or apolycarboxylate or a polyphosphonate salt. As further described below,the concerted action of the polyacid anion and the functional groups onthe silica surface promotes the irreversible adsorption of the proteincontaminants onto the solid phase.

The polycarboxylic acid has two or more carboxylate groups. Thepolycarboxylic acids may be saturated or unsaturated and may bealiphatic acids or aromatic acids, or cyclic saturated acids. Thepolycarboxylic acids may incorporate heteroatoms as part of the carbonchain between carboxylate functions or as substituent groups. Thepolycarboxylic acids may be substituted or unsubstituted and may belinear or branched chain.

The polycarboxylic acids may be saturated or unsaturated and may bealiphatic acids or aromatic acids, or cyclic saturated acids. Thepolycarboxylic acids may incorporate heteroatoms as part of the carbonchain between carboxylate functions or as substituent groups. Thepolycarboxylic acids may be substituted or unsubstituted and may belinear or branched chain.

The polycarboxylic acids may include, but are not limited to, saturatedand unsaturated aliphatic acids such as oxalic acid, malonic acid,succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid,azelaic acid, sebacic acid, maleic acid, fumaric acid, itaconic acid,glutaconic acid, citraconic acid, mesaconic acid and their derivatives,malic acid, tartaric acid, glutamic acid, galactaric acid. Aromaticacids include phthalic acid, isophthalic acid, terephthalic acid. Cyclicsaturated acids include camphoric acid. Some other acids may have threeor more carboxylate groups or incorporating heteroatoms: tricarballylicacid, trimesic acid and its isomers, mellitic acid and isocitric acid.

In one embodiment the polycarboxylic acid comprises a dicarboxylic acid.The dicarboxylic acid may be aliphatic, and may be unsubstituted. In oneembodiment the dicarboxylic acid is COOH—(CH₂)_(q)—COOH where q is inthe range of 4 to 6. In one embodiment the dicarboxylic acid is adipicacid or is pimelic acid. In one embodiment the dicarboxylic acid isadipic acid.

As an alternative to the polycarboxylic acids, analogous polyphosphonicacids may be used. Such phosphonic acids are typically analogues of theabove polycarboxylic acids such as linear alkane diphosphonic orhydroxyalkane diphosphonic acids. One such acid is etidronic acid, whichis a diphosphonic acid.

In one embodiment the polyacid is present at a concentration up to 200mM. The removal of protein contaminants may be carried out at pH 4.5 topH 7.0. The presence of the polyacid may act as a buffer within this pHrange. pH may suitably be adjusted by an appropriate base such as analkali metal hydroxide such as sodium hydroxide or potassium hydroxide.

In one embodiment a composition for the removal of protein contaminantsfrom a solution containing target nucleic acid is disclosed. Thecomposition comprises (i) a polyacid, which comprises a polycarboxylicacid, a polyphosphonic acid or a polycarboxylate or polyphosphonatesalt; (ii) a solid phase having a surface which comprises afunctionalized silica.

The polyacid or salt thereof and the solid-phase are as describedherein.

The above composition may be applied directly to a compositioncontaining target nucleic acid and protein contaminants such as nucleicacid enzymes.

In one embodiment, a kit for the removal of protein contaminants from asolution containing target nucleic acid is disclosed. The kit comprises(i) a polyacid, which comprises a polycarboxylic acid, a polyphosphonicacid or a polycarboxylate or polyphosphonate salt; and (ii) a solidphase having a surface which comprises a functionalized silica.

The polyacid or salt thereof and the solid phase may be supplied asseparate components in a kit, optionally together with instructions fortheir use. Each component of the kit may be supplied in separatecontainers. The kit may comprise further components such as othersolutions for use in removal of the protein contaminants.

The polyacid or salt thereof and the solid phase are as describedherein.

In one embodiment a nucleic acid reaction kit is disclosed. The nucleicacid reaction kit may comprise a nucleic acid enzyme and a compositionfor the removal of protein contaminants as described herein.Alternatively, the nucleic acid reaction kit may comprise a nucleic acidenzyme for acting on a target nucleic acid and a solid phase having asurface which comprises a functionalized silica for the removal of thenucleic acid enzyme from a solution containing the target nucleic acid,where the functionalized silica bears anionic or neutral substituentgroups which are (i) polar, other than phenol, and/or (ii) comprise a C₁to C₃ alkyl chain.

The nucleic acid reaction kit may contain instructions for use. Eachcomponent may be supplied in a separate container. The nucleic acidenzyme and the solid-phase are described in further detail herein. Thisnucleic acid reaction kit may further comprise a polyacid or saltthereof. The polyacid or salt thereof may be supplied in a furthercontainer or may be present with the nucleic acid enzyme or the solidphase.

Additional reagents may be included in the protein removal composition,such as conservants, stabilizers, or additional reagents for removal ofother components coming from upstream reaction mixtures.

While not limited to a specific theory, it is thought that theinteraction between functionalized silica and protein mainly proceeds bymutual attraction of:

a) hydrophobic moieties that include methylene handles of cyanopropylgroups, capping alkyl groups when present on solid phase, and siloxanegroups on silica vs. hydrophobic patches on protein molecule; andb) ionized groups such as negatively charged terminal nitriles ofcyanopropyl groups and residual silanols on silica vs. positivelycharged amino acid residues on protein molecule.

The concerted action of dicarboxylic acid or other diacid anions andfunctionalized silica promotes the irreversible adsorption ofproteinaceous compounds onto the solid phase. For example, molecules ofaliphatic dicarboxylic acids, e.g., adipic acid, either in protonated orin deprotonated form, have in solution hydrophobic (containing methylenechain) and hydrophilic (containing carboxyls) halves because of itsoverall curved structure (Min et al., Chem. Eur. J. 16: 10373 (2010)).This is achieved by intramolecular hydrogen bonding of an OH donor ofone carboxyl group to the carbonyl oxygen of another carboxyl group,acting as an acceptor. Additionally, the functional groups immobilizedon the porous silica structures disrupt the extensive intermolecularhydrogen bonding network characteristic for the solutions ofdicarboxylic acids and favor the formation of intramolecular hydrogenbonding. This way a large fraction of acid molecules acquiresconsiderable dipole momentum and may act as ion-pairing reagents.

This property of diacids such as dicarboxylic acids may allow mutualattraction between sterically isolated, normally non-interacting aminoacid residues on the protein surface and active groups on the silicasurface by creating the bridges between them.

Larger numbers of interacting amino-acid residues may also destabilizethe tertiary structure of an active enzyme. The inventors have foundthat even if the silica microspheres containing the adsorbed enzyme arenot removed by centrifugation, usually no residual or reactivatableenzymatic activity is detected after incubation at conditions favoringreactivation, and this activity does not increase (as in the case ofreactivation) in time.

These findings show that the inventive Protein Removal Reagent (PRR)acts not only by enthalpic adsorption processes which includehydrophobic and electrostatic interaction, hydrogen bonding, dipoleinteractions and van der Waals forces, but by entropic processes too.Hydrophobic groups at silica surface and aliphatic chains ofdicarboxylic acids or other diacids disrupt the native hydrogen bondnetwork of water and nearby protein structures, mainly (anti)parallelbeta-structures responsible for the shape of native protein molecule.Carboxyl groups of dicarboxylic acids form non-native salt bridges withpositively charged protein side chains. These entropic processes areessentially irreversible and result in the decrease of the orderedconformation of bound proteins resulting in disruption of tertiarystructures, expulsion of interstitial water, release of counter-ionsresponsible for maintaining native structure and activity. Both theseprocesses yield inactive unfolded proteins fixed onto the silicasurface.

This attraction is much stronger than the chelating action of carboxylicacids alone. Whereas the affinity for divalent ions necessary foractivity of most enzymes is by orders of magnitude greater for EDTA,iminodiacetic acid, or nitrilotriacetic acid derivatives, thesecompounds have much lower efficiency in the composition when usedinstead of aliphatic dicarboxylic acid.

This attraction is much stronger than an interaction between adsorbentssuspended in pure water. Although all tested adsorbents bearing diversefunctional groups (sulfopropyl, cyanopropyl, hydroxy, etc.) did displayefficiency in enzymes absorption, the efficiency was lower as comparedto that characteristic to that of the inventive composition.

Treatment with an inventive composition is more efficient thantraditionally employed protein removal methods, such asthermoinactivation of enzymes by heat in the excess of chelating agent(EDTA), or commercially available products for protein removal. Enzymesthat are not amenable to heat inactivation, e.g., some restrictionendonucleases, are readily removed by the inventive compositions.Residual dicarboxylic acid ions remaining in the reaction mixture aftertreatment did not interfere with the majority of downstreamapplications. No chaotropic or other active compounds are used in theinventive (or high concentrations of less active compounds, like salts,detergents or and water-soluble organic materials) unlike in knownformulations such as U.S. Pat. No. 7,115,719, thereby making theinventive compositions and methods environmentally safe.

Commercial products for protein removal currently available, such asDNAfree™ Kit (Ambion, McCormick, Analyt. Biochem. 181: 66 (1989);DNA-free™ Kit Ambion® Manual #1907M revision F. Jul. 9 2009) are byorders of magnitude less efficient than the inventive compositions.

Protein removal with a protein removal reagent (PRR) typically involvesa) admixing the inventive composition with nucleic acid containingsolution, in one embodiment at a ratio 1:10 or ratios dependent on theprotein quantity present in the solution, e.g. one μL PRR decreases theactivity of 0.5 unit of wild-type DNase I, or E. coli RNase I, orvarious restriction enzymes beyond the detection limit. Protein sorptiononto PRR is achieved by mixing the resulting suspension by vortexing forseveral seconds; b) suspension centrifugation immediately after admixingwith PRR and recovery of supernatant containing protein-free nucleicacid solution. The method may be performed at room temperature and doesnot require additional incubation time for protein binding.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows complete DNase I removal by synergistic action of CN-silicaand dicarboxylic acid in Protein Removal Reagent.

FIG. 2 shows examination of various dicarboxylic acids in combinationwith CN-silica for DNase I removal.

FIG. 3 demonstrates irreversible adsorption of DNase I by ProteinRemoval Reagent.

FIG. 4 demonstrates irreversible adsorption of RNase I by ProteinRemoval Reagent.

FIG. 5 shows removal of FastDigest® Pstl restriction enzyme fromreaction mixture by Protein Removal Reagent.

FIG. 6 shows removal of Taq DNA polymerase from reaction mixture byProtein Removal Reagent.

FIGS. 7A-F show SDS-PAGE visualisation of proteins extraction by ProteinRemoval Reagent.

FIG. 8 shows RNA yields after Protein Removal Reagent treatment.

FIG. 9 shows compatability of DNase I and Protein Removal ReagentTreated Total RNA with Real-Time RT-PCR.

FIG. 10 shows efficiency of DNase I removal by Protein Removal Reagentcompared to commercially available analogs.

The invention is now described in further detail with reference to thefollowing Examples and related Figures.

EXAMPLE 1 PRR Composition Synergistic Action of Silica Adsorbent andDicarboxylic Acid Salts

Several versions of PRR were prepared by suspending cyanopropylderivatized silica (50% slurry) either in pure water or in buffercontaining dicarboxylic acid salt (Na-adipate or Na-suberate) at pH 5.0.Efficacy of prepared PRR samples was tested as their ability to removeDNase I from reaction mixture. One unit DNase I suspended in 20 μl 1×Reaction Buffer with MgCl₂ was treated with 2 μl PRR variant. Forcomparison, aliquots of the same reaction mixture had been treated withthe same volumes of pure Na-adipate or Na-suberate buffer withoutsilica. All samples were centrifuged and 20 μL of supernatant from eachsample were transferred to a fresh tube. One μg supercoiled pUC 19plasmid DNA was added to each tube and incubated for two h at 37° C. todetect residual DNase I activity. In parallel, DNase I activitycalibration was performed to allow evaluation of the DNase I amount insample from extent of pUC 19 DNA degradation. From 1 to 10⁻⁷ units DNaseI were added to 1 μg pUC19 DNA in 20 μl 1× Reaction Buffer with MgCl₂and incubated for two h at 37° C. to resemble experimental conditions.All samples were analyzed electrophoretically in 1% agarose gel in TAEbuffer and the gel was stained with ethidium bromide to visualize DNA.

FIG. 1 shows complete DNase I removal by synergistic action of CN-silicaand dicarboxylic acid in Protein Removal Reagent. Upper half: 1 unitDNase I in 20 μl of 1× Reaction Buffer with MgCl₂ was treated with 2 μlProtein Removal Reagent prepared by suspending CN-silica either in purewater (lanes 5, 6) or in buffer containing Na-adipate (lanes 7, 8) orNa-suberate (lanes 9, 10). For comparison, aliquots of the same reactionmixture had been treated with the same volumes of Na-adipate (lanes 1,2)or Na-suberate (lanes 3, 4) buffer without CN-silica. Two independentreactions were performed for each sample. After centrifugation 20 μlsupernatant was transferred to a fresh tube and 1 μg pUC19 DNA was addedto each tube and incubated for two h at 37° C. to test for residualDNase I activity. Reactions were analyzed on an agarose gel followed bygel staining with ethidium bromide. Lower half: DNase I were added to 1μg of pUC19 DNA in 20 μl of 1× Reaction Buffer with MgCl₂ and incubatedfor two h at 37° C. to test the dependence of DNA degradation on DNase Iunits at the conditions of the experiment. Two independent reactionswere performed for each DNase I amount: 1 u (lanes 1, 2), 10⁻² u (lanes3, 4), 10⁻⁴ u (lanes 5, 6), 10⁻⁶ u (lanes 7, 8), 10⁻⁷ u (lanes 9, 10)and no DNase I control (lanes 11, 12).

As shown in FIG. 1, dicarboxylic acid salts alone had no influence onDNase I activity as the pUC 19 DNA was fully degraded in these samples.There was only slight increase of linear DNA in samples treated withderivatized silica suspended in water, indicating that residual DNase Iactivity was lower by about five orders of magnitude compared to initialDNase I activity in the sample. Inclusion of adipate or suberate bufferin PRR formulation decreased residual DNase I level left in sample aftertreatment with PRR by one to two orders of magnitude further asindicated by undetectable DNase I activity in these samples.

EXAMPLE 2 PRR Composition: Performance of PRR Buffer Components

Several versions of PRR were prepared by suspending cyanopropylderivatized silica in buffers containing salts of various dicarboxylicacids. All dicarboxylic acids have been used as sodium salts at pH 5.0,and cyanopropyl silica has been used as an adsorbent. Each PRR wastested for protein removal efficacy from solution using DNase I as amodel enzyme.

Two μl of various PRR were used to remove 2 U of DNase I from 20 μl 1×Reaction Buffer with MgCl₂ followed by residual DNase I activity testingin supernatant with pUC 19 DNA, as described in the previous example.

FIG. 2 shows examination of various dicarboxylic acids in combinationwith CN-silica for DNase I removal. 50% suspension of CN-silica inbuffer of dicarboxylic acid (2 μl) was used for removal of DNase I (2unit in 20 μl of 1× Reaction Buffer with MgCl₂). After centrifugation 20μl supernatant was transferred to a fresh tube and 1 μg pUC19 DNA wasadded and incubated for two h at 37° C. to test for residual DNase Iactivity. Reactions were analyzed on an agarose gel and stained withethidium bromide. Two independent reactions were performed for eachdicarboxylic acid tested: succinate (lanes 1, 2), iminodiacetate (lanes3, 4), glutarate (lanes 5, 6), glutamate (lanes 7, 8), adipate (lanes 9,10), pimelate (lanes 11, 12), suberate (lanes 13, 14). Control ofsupercoiled pUC 19 DNA was loaded in lanes 15 and 16.

Results presented in FIG. 2 show that all tested salts of dicarboxylicacids are suitable for PRR preparation. The longer the methylene chainof aliphatic dicarboxylic acids, the better performance of the PRR, withthe exception of very short-chain acids such as succinate and oxalate(data not shown). The latter exception may be explained by the generalinhibitory power of short dicarboxy acids on many enzymes, illustratedby the number of enzymes inhibited by each dicarboxylic acid referencedin the BRENDA database (http://www.brenda-enzymes.org), therefore theyare less suitable as PRR buffer components:

Number of CH₂ groups Number of between carboxylates Dicarboxylic acidinhibited enzymes 0 oxalic 119 1 malonic 63 2 succinic 115 3 glutaric 254 adipic 10 5 pimelic 3 6 suberic 0

Some other organic acids that showed satisfactory performance in thecapacity of PRR buffer component have no practical utility within thescope of the disclosed invention as they are known as strong inhibitorsof many enzymes, e.g., aurintricarboxylic acid (21 enzyme inhibited), orcitric acid (203 enzymes inhibited).

Better performance of longer aliphatic molecules is consistent with thepresumption that dipole structure of aliphatic dicarboxylic acids insolution enables their action as ion-pairing agents and thus facilitatesinteractions between sterically isolated functional groups on proteinsurfaces and those on adsorbent surfaces. Experimental data alsodemonstrate that introduction of charged functional groups decrease theperformance of buffer component. This is evident from the behavior oftwo pairs of acids: succinic vs iminodiacetic (two methylenes), andglutaric vs glutamic (2-aminoglutaric, three methylenes). In both casesnitrogen-containing group decreased the hydrophobicity of methylenechain, and the molecule acquires purely zwitterionic structure devoid ofhydrophobic face and thus cannot longer act as an ion-pairing reagent.

EXAMPLE 3 Irreversibility of PRR Action

Types of protein interaction with PRR was tested using DNase I and E.coli RNase I enzymes as models. Reaction mixtures containing either 1 UDNase I in 20 μL 1× Reaction Buffer with MgCl₂ or 20 U RNase I in 20 μL1× FastDigest® Buffer were treated with 2 μL or 1 μL of PRR,respectively. Both supernatant and pellet after centrifugation weresaved.

Supernatants were complemented with 1/10 volume of corresponding 10×enzyme reaction buffer to reestablish original reaction conditions,while pellets were re-suspended in the original reaction volumes of 1×corresponding enzyme reaction buffers.

In case the enzymes were inhibited by soluble components of PRR, e.g.,by complexation of cofactor metal ions, the addition of reaction buffercomponents would reconstitute reaction conditions needed for enzymereactivation and activity. This effect, if present, could be potentiallydangerous for downstream applications. In case the protein binding byCN-silica was somewhat reversible the released amounts of enzymes couldbe recovered after some period of time. This effect could also bepotentially dangerous if there was delay before centrifugation or/andsupernatant transfer or occasionally some silica particles aretransferred together with the supernatant to a fresh tube.

Supernatants and re-suspended pellets were incubated at room temperaturegenerally favoring protein refolding and renaturation. After fixedperiods of time, aliquots were taken from the samples and tested forenzymatic activities. DNase I activity was assayed with pUC 19 DNA asdescribed in Example 1. RNase I activity was measured by sampleincubation with [³H]-RNA and subsequent counting of radioactivityreleased into acid-soluble fraction. RNase I samples were supplementedwith 200 μg [³H]-RNA and incubated for 30 min at 37° C. Subsequently twovolumes of cold 10% TCA were added and samples were kept for 15 min inan ice bath, followed by ten min centrifugation at 14 000 rpm.Supernatant was transferred to a scintillation vial containing 5 mlscintillation cocktail Rotiszint® eco plus (Roth) and radioactivity wascounted with a Beckman LS 1801 scintillation counter. Recovered RNase Iactivity was recalculated in % of RNase I activity in control samplewithout any treatment.

DNase I assay results are shown in FIG. 3. RNase I assay results areshown in FIG. 4.

FIG. 3 demonstrates irreversible adsorption of DNase I by ProteinRemoval Reagent. One unit of DNase I in 20 μl 1× Reaction Buffer withMgCl₂ was treated with 2 μl PRR. After centrifugation the supernatantwas complemented with 1/10 volume of 10× Reaction Buffer with MgCl₂,while the pellet was re-suspended in 20 μl 1× Reaction Buffer withMgCl₂. Enzyme was allowed for to renaturate and/or dissociate fromadsorbent for 3 min, 1.1 h, 3 h, or 93 h (lanes 2, 4, 6, 8 forsupernatant samples and lanes 3, 5, 7, 9 for pellet samplesrespectively) at room temperature. To evaluate recovered DNase Iactivity 1 μg pUC19 DNA was added and incubated for two h at 37° C. Allreactions and control of intact pUC 19 DNA (lane 1) were analyzed on anagarose gel and stained with ethidium bromide.

FIG. 4 demonstrates RNase I by Protein Removal Reagent. Twenty U ofRNase I in 20 μL FastDigest® buffer was treated with 1 μL PRR. Aftercentrifugation the supernatant was complemented with 1/10 volume 10×FastDigest® buffer, while the pellet was re-suspended in 20 μl of 1×FastDigest® buffer. After incubation for 3 min, 1.1 h, 3 h, or 93 h atroom temperature, 200 μg [³H]-RNA was added and incubated for 30 min.After precipitation with TCA radioactivity released into acid-solublefraction was counted. Recovered RNase I activity was recalculated in %of RNase I activity without treatment with PRR. Triangles: residualRNase I activity extracted from adsorbent with 1× FastDigest® reactionbuffer. Circles: RNase I activity left in supernatant.

In both cases only traces of activity of tested enzymes were detected.Because no increase of residual enzyme activity was detected in thesupernatant after prolonged incubation, it was inferred that no enzymereactivation occurred, i.e., the protein was fully removed fromsolution. Even if the silica microspheres containing the adsorbed enzymewere suspended in reaction buffer resembling the situation if silicawith adsorbed protein is not removed by centrifugation, no residual orreactivatable enzymatic activity was detected after prolonged incubationat conditions favoring reactivation. This indicated that the proteinbinding by PRR was fully irreversible.

EXAMPLE 4 Efficiency of PRR in Removal of Various Proteins

Universal action of several PRR versions in removing various proteinsfrom reaction mixtures was tested using enzymes that, in a molecularbiology research workflow, are most commonly removed from reactionmixtures before taking their substrate into the new subsequent reaction.

PRR was prepared by suspending cyanopropyl derivatized silica (50%slurry) either in pure water or in buffer containing dicarboxylic acidsalt (Na-adipate or Na-suberate) at pH 5.0.

Functional Removal of Restriction Enzymes

Restriction enzymes are often used in manipulations with DNA and, as arule, they must always be inactivated after DNA digestion. This isusually achieved by enzyme denaturation at high temperatures. However,many restriction enzymes, both thermophilic and mesophilic, withstandheat treatment, so tedious protocols of DNA purification by organicsolvents must be applied. Functional removal of restriction enzymes fromreaction mixtures by PRR is illustrated using FastDigest® Pstlrestriction enzyme as an example; several other enzymes have been testedas well. Although mesophilic, this particular enzyme is not inactivatedby heat.

The experiment was performed as follows: 20 μL reaction mixturecontaining 1× FastDigest® reaction buffer and 1 FDU of enzyme wasprepared. Two μL PRR reagents, in water or Na-suberate, were added. Theresulting mixtures were mixed by vortex mixing, the silica was pelleted(spun down), and supernatants were saved into fresh tubes. One μg Aphage DNA was added to all reaction mixtures. Control samples containingonly water or suberate buffer (without silica) were incubated for fivemin, while PRR treated samples were incubated for two h at 37° C.Results were analyzed by DNA electrophoresis.

FIG. 5 shows removal of FastDigest® Pstl restriction enzyme from areaction mixture by Protein Removal Reagent. One μl FastDigest® Pstl in20 μL 1× FastDigest® buffer was treated with 2 μL CN-silica suspensionin water (lanes 5, 6), or in 100 mM Na-suberate (lanes 7, 8). Aftercentrifugation supernatants were supplemented with 1 μg A phage DNA andincubated for two h at 37° C. to test residual restriction enzymeactivity. Control samples were prepared by adding only water (lanes 1,2) or Na-suberate buffer (lanes 3, 4) to Pst I reaction mixture and wereincubated for five min at 37° C. All reactions were prepared induplicate and with intact A phage DNA control (lanes 9, 10) wereseparated by electrophoresis in agarose gel with ethidium bromide.

Results in FIG. 5 show that 2 μL PRR was able to completely remove 1 FDUof restriction enzyme Pstl. Other FD restrictions enzymes tested, EcoRV,BgIII, were successfully removed as well. Functional removal ofpolymerases

Functional removal of polymerases by PRR was tested using Thermusaquaticus DNA polymerase. This enzyme, being thermophilic, is impossibleto inactivate by heat.

The experiment was performed as follows: 50 μL reaction mixturecontaining 1× Taq Buffer with KCl (10 mM Tris-HCl (pH 8.8), 50 mM KCl,0.08% Nonidet P40, 1.5 mM MgCl₂) and 1.25 U enzyme was prepared. Five μLPRR reagents, in water or Na-adipate or Na-suberate, were added,mixtures were vortexed, silica was pelleted (spun down) and supernatantswere saved in fresh tubes. Control samples containing only water ordicarboxylic acid buffer (without silica) were prepared. Polymeraseactivity was determined in all samples. Five μL supernatant or controlsample was combined with 45 μL mix containing 67 mM Tris-HCl (pH 8.8),6.7 mM MgCl₂, 1 mM DTT, 50 mM NaCl, 0.1 mg/mL BSA, 0.25 mg/mL activatedsalmon sperm DNA, 0.2 mM dNTP, and 0.37 MBq/mL tritium-labeled dTTP.Reactions were incubated for 30 min at 70° C. Forth μl reaction wasspotted on DE-81 paper disc. Paper discs were washed four times for tenmin with 7.5% Na₂HPO₄×10 H₂O, rinsed twice with water and once withacetone, dried, placed into a scintillation vial containing 5 ml ofscintillation cocktail Betaplate Scint (Perkin Elmer), and counted forradioactivity with Beckman LS 1801 scintillation counter. Residualpolymerase activity (%) was calculated with respect to polymeraseactivity in control sample with water (without silica).

FIG. 6 shows removal of Taq DNA polymerase from reaction mixture byProtein Removal Reagent. 1.25 U Taq DNA polymerase in 50 μL of 1× TaqBuffer with KCl was treated with 5 μL of CN-suspension in water (columnWS) or in 100 mM Na-adipate (column AW) or Na-suberate (column SW).After centrifugation 5 μl of supernatants was added to 45 μl ofpolymerase activity measurement mixture (67 mM Tris-HCl (pH 8.8), 6.7 mMMgCl₂, 1 mM DTT, 50 mM NaCl, 0.1 mg/mL BSA, 0.25 mg/mL activated salmonsperm DNA, 0.2 mM dNTP, 0.37 MBq/mL [³H]-dTTP) and incubated for 30 minat 70° C. Taq activity was calculated from radioactivity adsorbed onDE-81 filters. Control samples were prepared by addition of only wateror 100 mM Na-adipate (column AB) or Na-suberate buffer (column SB) toTaq I reaction mixture and polymerase activity was measured as describedabove. Residual Taq DNA polymerase activity was recalculated in % ofpolymerase activity in control sample with water. All displayed resultsare the mean values of at least three experiments with error bars of onestandard deviation.

The results shown in FIG. 6 demonstrated that after treatment withcyanopropyl silica suspension alone in pure water, polymerase activitywas reduced by about two orders of magnitude. Enzyme removal withcyanopropyl silica in water was significant; concerted action ofcyanopropyl derivatized silica and dicarboxylic acid salt resulted insubstantially complete polymerase removal as the measured polymeraseactivities were lower than the experimental error margins.

Physical Removal of Various Proteins from Reaction Mixtures

Analytical protein electrophoresis demonstrated that the principle ofPRR action was based solely on the physical removal of proteins fromreaction mixtures. Experiments were performed with a variety of proteinsextracted with PRR from their conventional reaction mixtures.Experiments were performed as follows: 20 μL of the reaction mixturecontaining conventional enzyme reaction buffer and quantity of enzymeusually employed in routine molecular biology procedures were prepared.One-tenth volume of PRR reagent in water or Na-adipate or Na-suberatewere added, mixtures were mixed by vortex mixer, silica was pelleted(spun down) and supernatants were saved in fresh tubes. Control samplescontaining only water or dicarboxylic acid buffer (without silica) werealso prepared. Supernatant and control samples were supplemented with 5μL 4× DualColor™ Protein Loading Buffer containing DTT. Silicaprecipitates were suspended in 20 μL 1× DualColor™ Protein LoadingBuffer containing DTT. All samples were incubated at 100° C. for tenmin, analyzed by SDS-PAGE with the gel stained with PageBlue™ ProteinStaining Solution or PageSilver™ Silver Staining Kit (Taq polymerase andDNase I gels).

The following proteins were tested in these experiments (quantity ofprotein in 20 μL reaction mixture): 2 μg bovine serum albumin (˜70 kDa)in water, 1 unit DNase I (˜30 kDa) in 1× Reaction Buffer with MgCl₂, 1unit FastAP™ alkaline phosphatase (˜40 kDa) in 1× FastAP Buffer (BSAincluded), 1 FDU FastDigest® Pstl restriction nuclease (˜40 kDa) in 1×FastDigest® Buffer (BSA included), 0.5 u Taq DNA polymerase (˜100 kDa)in 1× Taq Buffer with KCl, and 10 units E. coli RNase I (˜25 kDa) in 1×FastDigest® Buffer (BSA included).

FIGS. 7A-F show SDS-PAGE visualisation of proteins extraction by ProteinRemoval Reagent. 20 μL of the reaction mixture containing conventionalenzyme reaction buffer and quantity of enzyme usually employed inroutine molecular biology procedures were prepared and treated with 2 μLof CN-silica suspension in water or in 100 mM Na-adipate or Na-suberate.After centrifugation, supernatants were supplemented with 5 μl of 4×DualColor™ Protein Loading Buffer containing DTT (lanes 4, 5, 6 for CNsilica in water, Na-adipate and Na-suberate respectively), while thepellet was re-suspended in 20 μl of 1× DualColor™ Protein Loading Buffercontaining DTT (lanes 7, 8, 9 for CN silica in water, Na-adipate andNa-suberate respectively). Control samples were prepared by addition ofonly water (lane 1) or 100 mM Na-adipate (lane 2) or Na-suberate (lane3) buffer to enzymes and later supplemented with 5 μl of 4× DualColor™Protein Loading Buffer containing DTT for electrophoresis. All sampleswere denatured at 100° C. for 10 min and analyzed by SDS-PAGE andstained with PageBlue™ Protein Staining Solution except DNase I gel (B)and Taq DNA polymerase gel (E) stained with PageSilver™ Silver StainingKit. PageRuler™ Unstained Broad Range Protein Ladder (Thermo Scientific)was loaded in last M lane: 250, 150, 100, 70, 50, 40, 30, 20, and 15 kDabands were visible. The following proteins were tested in thisexperiment: (A) 2 μg of bovine serum albumin, (B) 1 unit of DNase I, (C)1 unit of FastAP™ alkaline phosphatase, (D) 1 FDU of FastDigest® Pstlrestriction nuclease, (E) 0.5 u of Taq DNA polymerase and (F) 10 unitsof E. coli RNase I. BSA band (˜70 kDa) from reaction buffer along withenzymes was visible in C, D and E gels.

The electrophoresis results were very similar for all proteins tested.The results clearly demonstrated that after treatment with PRR noprotein was left in the sample. Even if BSA was present in enzymereaction buffer (FIG. 7C, FastAP buffer and D, F, FastDigest® Buffer)PRR was able to remove enzyme together with BSA except when cyanopropylsilica suspension in water was used for FastAP™ alkaline phosphatase(FIG. 7C) or RNase I (FIG. 7F) extraction where a faint BSA band wasstill visible. However dicarboxylic acid salt inclusion in silicasuspension enabled physical removal of all tested proteins from reactionmixtures. Detergent included in Taq buffer had no interference forpolymerase extraction with all PRR tested. The dicarboxylic acid saltbuffers alone had no effect on the physical integrity of tested proteinsas these samples are identical to water samples. The protein binding toPRR was so strong that even very aggressive extraction conditions,boiling of suspended silica in sample buffer containing strong detergentSDS, failed to liberate the major portion of the protein adsorbed ontosilica.

In summary, the data showed that 2 μL PRR was able to bind irreversiblyand remove from reaction about 2 μg protein down to the quantityundetectable by SDS-PAGE. Although cyanopropyl silica microspheres alonephysically adsorb protein almost on par with the PRR suspended in thebuffer of dicarboxylic acid salt, they were less efficient in removingresidual functional activity of enzymes as was shown in the previouslydescribed examples, summarized in the table below:

Residual activity after PRR Enzyme treatment quantity in Volume ofCN-silica in Activity reaction reaction CN-silica in dicarboxylicmeasurement Enzyme mixture, U mixture*, μL water acid buffer methodDNase I 1 20 <10⁻⁵ u undetectable Functional assay - supercoiled plasmidDNA degradation RNase I (E. coli) 20 20 <0.03% <0.001% Functional assaywith [³H]-RNA - counting of radioactivity released into acid- solublefraction Phosphatase 1 20 —  0-0.4% Functional assay - FastAP ™spectrophotometrical detection of p-NPP hydrolysis FastDigest ®   1FDU*** 50 none none Functional assay - λ EcoRV** DNA digestionFastDigest ® 1 FDU 50 none none Functional assay - λ PstI DNA digestionFastDigest ® 1 FDU 50 none none Functional assay - λ BglII DNA digestionTaq DNA 1.25 50     1%  <0.03% Functional assay - polymeraseincorporation of nucleotides into a polynucleotide fraction (absorbed onDE-81) *in all cases 1/10 volume of PRR was added to reaction mixturesfor enzyme removal **results not presented **FDU—FastDigest units

EXAMPLE 5 Nucleic Acids Yields after PRR Treatment

Many molecular biology research workflows may be described as aconsecutive series of reactions, where certain starting nucleic acidacts as a substrate for several subsequent enzymatic reactions. Oftenthe next enzymatic reaction requires removal of the preceding enzymefrom the reaction mixture. In such a workflow it is important thatmethods used for enzyme removal do not have negative effect on theconcentration of the nucleic acid remaining in the reaction mixture,i.e., it is desirable that enzyme removal should occur without anyconcurrent loss of the substrate nucleic acid. The effect of PRRtreatment on the concentration of nucleic acid remaining in the reactionmixture was tested using synthetic RNA as a control and DNase I as aprotein for removal (FIG. 8).

FIG. 8 shows RNA yields after Protein Removal Reagent treatment.Solutions of 100 ng synthetic 2 kb RNA in 20 μL 1× Reaction Buffer withMgCl₂ were treated with 4 μL of various PRRs (all reactions wereprepared in duplicate) made as CN-silica suspension in differentbuffers—50 mM adipate, pH 5 (lanes 3, 4), 50 mM suberate, pH 5 (lanes 7,8), 20 mM suberate, pH 5 (lanes 9, 10), 40 mM suberate with 10 mMammonium sulfate, pH 5 (lanes 11, 12) and 40 mM non-buffered ammoniumsulfate (lanes 13, 14). PRR prepared from phenyl-silica in 50 mMsuberate (lanes 3, 4) known to bind nucleic acids was included as anegative control. Supernatants together with untreated RNA (lanes 1, 2,15, 16) were analyzed by agarose gel electrophoresis for the remainingamount of RNA.

Solutions of 100 ng synthetic 2 kb RNA in 20 μL of 1× Reaction Bufferwith MgCl₂ were treated with 4 μL of various PRRs which were made as 50%CN-silica suspension in different buffers: 50 mM adipate, pH 5.0, 50 mMsuberate, pH 5.0, 20 mM suberate, pH 5.0, 40 mM suberate with 10 mMammonium sulfate, pH 5.0 and 40 mM non-buffered ammonium sulfate.Supernatants were analyzed by gel electrophoresis (1% agarose in TAEwith ethidium bromide) for remaining amount of RNA. It is evident thatPRRs based on 50 mM dicarboxylic acids salts buffers did not bind RNA,while RNA retention by PRR increased with decreasing bufferconcentrations and slightly increased with increased overall ionicstrength. Phenyl derivatized silica known to bind nucleic acids wasincluded for comparison as negative control.

EXAMPLE 6 PRR Treatment Did not Interfere with Downstream Reactions(RT-QPCR)

One of the most frequently performed procedures in molecular biology isDNA synthesis from small RNA quantities present in samples of variousnature. Typically, this procedure is executed as a consecutive set ofreactions following RNA purification, including DNase I treatment,reverse transcription, and qPCR where the residual DNase I must beremoved prior to reverse transcription. It is important that DNase Iremoval procedures and reagents used in this process have no negativeimpact on nucleic acid integrity and yield during reverse transcriptionand, as no additional purification is performed after RT, during theqPCR step.

qPCR is a very sensitive test for evaluation of nucleic acid yield afterPRR treatment. The RT-qPCR procedure may be carried out in aquantitative fashion and nucleic acid yield may be measured in a broadrange of concentrations starting from minute quantities of the initialRNA material.

The experiment was performed as follows: 10-fold serial dilutions from100 ng to 1 μg of HeLa total RNA were treated with 1 U of DNase I in 20μl 1× Reaction Buffer with MgCl₂ for 30 min at 37° C. and then the DNaseI was removed with 2 μl of PRR. Four μl supernatant was used for cDNAgeneration with the Maxima™ First Strand cDNA Synthesis Kit and GAPDH(glyceraldehyde-3-phosphate dehydrogenase) cDNA was detected insubsequent qPCR with the Maxima™ SYBR Green qPCR Master Mix (2×), ROXSolution provided. Untreated RNA dilutions in 1× Reaction Buffer withMgCl₂ were used in RT-qPCR as controls.

FIG. 9 shows that DNase I and Protein Removal Reagent treated total RNAwas compatible with Real time PCR (RT-PCR). Ten-fold serial dilutionsfrom 100 ng to 10 μg (curves from left to right respectively) of HeLatotal RNA were treated with 1 U DNase I in 20 μl 1× Reaction Buffer withMgCl₂ for 30 min at 37° C. and then the DNase I was removed with 2 μl ofPRR. Four μl supernatant was used for cDNA generation with the Maxima™First Strand cDNA Synthesis Kit, and GAPDH cDNA was detected insubsequent qPCR with the Maxima™ SYBR Green qPCR Master Mix (2×), ROXSolution provided (dark grey curves). Untreated RNA dilutions in 1×Reaction Buffer with MgCl₂ were used in RT-qPCR as controls (light greycurves).

Results in FIG. 9 demonstrated that PRR treated RNA was compatible withRT-qPCR and PRR did not extract any RNA, as PRR treatment had nonegative effect on RNA yields even at the lowest quantity of RNA used inthe experiment.

EXAMPLE 7 Comparison of PRR Efficiency with Commercially AvailableAnalogs

PRR efficiency was tested using pancreatic DNase I as a model enzyme forremoval efficacy from a solution containing RNA. PRR efficiency wascompared with heat inactivation and the following commercial analogs:

-   i) Ambion® TURBO DNA-free™ Kit (Invitrogen, Inc.), used per the    manufacturer's recommendations: 2 units of TURBO DNase were removed    using 2 μL of DNase removal reagent from the kit-   ii) RTS DNase™ Kit (MoBio Laboratories, Inc.), used per the    manufacturer's recommendations: 4 units of DNase were removed using    2 μL of DNase removal reagent from the kit-   iii) DNase I (Stratagene) and StrataClean Resin (Stratagene), 10    units DNase I were removed using StrataClean resin in three    successive extraction rounds per the manufacturer's recommendations-   iv) recombinant DNase I (Takara Bio Group) removed using QuickClean™    Enzyme Removal Resin (Clontech, part of Takara Bio Group), 1 unit    Recombinant DNase I was removed using QuickClean™ Enzyme Removal    Resin in three successive extraction rounds per the manufacturer's    recommendations.

Primary reaction mixtures were prepared as follows: 20 μL of solutioncontaining 1 μg RNA (200 b long) and 0.1 μg DNA (HindIII fragments of Aphage DNA) in appropriate DNase I reaction buffer had been supplementedwith 1 unit of Thermo Scientific DNase I in PRR test and heatinactivation experiments, or with various other DNases as describedabove.

After 30 min incubation at 37° C., Thermo Scientific DNase I wascaptured by adding 2 μL of PRRHeat inactivated samples supplemented with2 μL of 50 mM EDTA and heated at 65° C. for 10 min. Other DNases wereremoved using corresponding removal agents recommended by DNase supplier(or available from the same supplier in the case of standalone DNase) asdescribed above.

All samples were mixed thoroughly and centrifuged at 2400 rpm for 30sec. Final supernatants were saved in another set of tubes. Allreactions were performed in duplicate and 2 μL of water was added to onereplicate and 2 μL of corresponding 10× DNase buffer concentrates wasadded to another replicate to reconstitute DNase reaction conditionsdesignated as renaturation controls. Renaturation controls were includedto assess completeness of enzyme removal by ruling out possibleinhibitory effects of divalent metal depletion by removal withDNase/protein removing resins or by making complexes with buffercomponents of resins.

Subsequently, 2 μL of solution containing 1 μg supercoiled pUC19 plasmidwere added to each sample to provide a substrate for remaining DNase Ileft unbound and samples were incubated for two h at 37° C. (secondaryreaction mixtures). All samples had been analyzed electrophoretically in1% agarose gel in TAE buffer with the gel stained with ethidium bromideto visualize DNA (FIG. 10).

RNA was treated with DNase and then the DNase was removed by followingthe procedure for routine DNA removal as described. RNA samplescontaminated with DNA were prepared by mixing 1 μg RNA (200 b long) with0.1 μg DNA (HindIII fragments of A phage DNA) in 20 μL volume. The RNAsample was incubated for 30 min at 37° C. with 1 unit DNase I (ThermoScientific) in 1× Reaction Buffer with MgCl₂ to degrade DNA, and 2 μl ofPRR (CN-silica in 50 mM Na-adipate) was used to remove DNase I from thesample. The samples of commercial kits or resins were treated withcorresponding DNase and then the DNase was removed per the manufacturersrecommendations. The thermo-inactivated sample was incubated for 30 minat 37° C. with 1 unit of DNase I (Thermo Scientific) in 1× ReactionBuffer with MgCl₂ and after that was supplemented with 2 μL of 50 mMEDTA and heated at 65° C. for ten min to inactivate DNase I. All sampleswere prepared in duplicates. After centrifugation 2 μL of water wasadded to the supernatant of one replicate and 2 μl of corresponding 10×DNase buffer to another replicate. Subsequently, 2 μL of solutioncontaining 1 μg supercoiled form of pUC19 plasmid was added to eachsample to provide a substrate for remaining DNase I and samples wereincubated for two h at 37° C. All samples and pUC 19 DNA control wereanalyzed electrophoretically. Lanes M—GeneRuler™ Express DNA Ladder,100-5000 bp; Lane 1—supercoiled plasmid pUC19 DNA control; lanes 2,3—samples treated with DNase I (Thermo Scientific) and PRR without andwith extra addition of Reaction Buffer with MgCl₂ respectively; lanes 4,5—samples treated with Ambion®TURBO DNA-free™ Kit (Life Technologies)without and with extra addition of TURBO DNase I Buffer respectively;lanes 6, 7—samples treated with RTS DNase™ Kit (MoBio Laboratories)without and with extra addition of RTS DNase Buffer respectively; lanes8, 9—samples treated with DNase I (Stratagene) and StrataClean Resin(Stratagene) in three extraction cycles without and with extra additionof DNase I Buffer respectively; lanes 10, 11—samples treated withRecombinant DNase I (TAKARA BIO Inc.) and QuickClean™ Enzyme RemovalResin (Clontech) in three extraction cycles without and with extraaddition of DNase I Buffer (TAKARA B10) respectively; lanes 12,13—thermo-inactivated samples without and with extra addition ofReaction Buffer with MgCl₂, respectively.

DNase removal by PRR treatment was more complete than that by commercialanalogs or by DNase I thermoinactivation. The PRR action uses theirreversible denaturation and fixation of inactive protein on the solidphase instead of chelation of stabilizing enzyme cofactor metal ions.Supplementation of purified supernatant with divalent metal ions did notrestore enzymatic activity.

Although StrataClean resin shows similar final efficiency of DNaseextraction as PRR, StrataClean requires three successive extractionstages, while PRR needs only single extraction round to achieve completeDNase I removal. This repeated extraction inadvertently results insignificantly reduced RNA yield. StrataClean is believed to use a resinwhich has surface phenol groups.

While EDTA/heat inactivation results in complete DNase I inactivation,enzyme is not physically removed from reaction mixture. Uponreactivating conditions after addition of 1× Reaction Buffer with MgCl₂DNase I had reactivated to a degree sufficient for complete digestion ofDNA during secondary reaction. Heat treatment could also damage RNA,particularly if EDTA-unbound divalent metal ions are left.

Both TURBO® DNA-free™ DNase Treatment and Removal Reagents kit and RTSDNase™ Kit showed incomplete removal of their respective DNases.Although such DNase removal level may be sufficient for certainapplications, it may be detrimental for some more sensitive downstreamprocedures; QuickClean™ resin showed significantly lower ability toremove DNase even when three successive extraction rounds were deployed.

What is claimed is:
 1. A composition for removing protein contaminantsfrom a solution containing a target nucleic acid, the compositioncomprising (i) a polyacid comprising a polycarboxylic acid, apolyphosphonic acid, or a polycarboxylate or polyphosphonate salt; and(ii) a solid phase comprising a functionalized silica surface.
 2. Thecomposition of claim 1 where the solid phase comprises silica.
 3. Thecomposition of claim 1 where the solid phase comprises particles.
 4. Thecomposition of claim 3 where the particles are substantially sphericaland have a diameter in the range of from 3 μm to 15 μm.
 5. Thecomposition of claim 3 where the particles have pores with a diameter offrom 10 nm to 100 nm.
 6. The composition of claim 1 where thefunctionalized silica bears anionic or neutral substituent groups thatare (i) polar, other than phenol, and/or (ii) comprise a C₁ to C₃ alkylchain.
 7. The composition of claim 6 where the substituent groups areselected from cyanoalkyl groups (CN—(CH₂)_(n)—) where n is an integer ofat least 3; short chain alkyl groups (CH₃—(CH₂)_(m)—) where m is 0, 1,or 2; sulfoalkyl groups (HSO₃—(CH₂)_(l)—) where I is an integer in therange 2 to 6; alkanoyl groups (CH₃—(CH₃)_(p)—CO—O—) where p is 0, 1, or2; a diol ((OH)₂—CH—(CH₂)_(r)—) or hydroxyl OH—(CH₂)_(r)— where r iszero or an integer in the range 1 to 5, or mixed diolCH₂OH—CHOH—(CH₂)_(t)—; a cyclohexyldiol C₆H₉(OH)₂—(CH₂)_(t)—;alkylhalides Hal-(CH₂)_(t)—, tosylhalides SO₂Hal-C₆H₄—(CH₂)_(t)—, tosylSO₃H—C₆H₄—(CH₂)_(t)—, or alkylphenylhalidesHal-(CH₂)_(u)—C₆H₄—(CH₂)_(t)— where Hal is halogen, t is 2, 3, or 4, andu is 0, 1, or
 2. 8. The composition of claim 7 where the substituentgroups are cyanopropyl groups.
 9. The composition of claim 1 where thesurface of the solid phase further comprises silane groups of generalformula —O—Si—R₃ where each R is independently C₁-C₃ alkyl.
 10. Thecomposition of claim 9 where each R is methyl.
 11. The composition ofclaim 1 where the polyacid comprises a polycarboxylic acid.
 12. Thecomposition of claim 11 where the polycarboxylic acid comprises adicarboxylic acid.
 13. The composition of claim 12 where thedicarboxylic acid is aliphatic.
 14. The composition of claim 12 wherethe dicarboxylic acid is unsubstituted.
 15. The composition of claim 14where the dicarboxylic acid is COOH—(CH₂)_(q)—COOH where q is from 4 to6.
 16. The composition of claim 15 where the dicarboxylic acid is adipicacid.
 17. The composition of claim 1 where the polyacid is present at aconcentration up to 200 mM.
 18. The composition of claim 1 buffered at apH from 4.5 to 7.0.
 19. A nucleic acid reaction kit comprising a nucleicacid enzyme for acting on a target nucleic acid, a compositioncomprising (i) a polyacid comprising a polycarboxylic acid, apolyphosphonic acid, or a polycarboxylate or polyphosphonate salt; and(ii) a solid phase comprising a functionalized silica surface, andinstructions for using the kit to remove protein contaminants from asolution containing the target nucleic acid.
 20. A method for removingprotein contaminants from a solution containing target nucleic acid, themethod comprising contacting a solution containing target nucleic acidand protein contaminants with a solid phase having a surface comprisingfunctionalized silica bears anionic or neutral substituent groups whichare (i) polar, other than phenol, and/or (ii) comprise a C₁ to C₃ alkylchain. capable of binding protein under conditions that the proteincontaminants bind to the solid phase to form a treated solution; andseparating the treated solution from the solid phase, where thefunctionalized silica bears anionic or neutral substituent groups thatare (i) polar, other than phenol, and/or (ii) comprise a C₁ to C₃ alkylchain.